Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum

A fast and sensitive LC–MS/MS method for the quantitative analysis of seven steroid hormones in 150 μl of human serum was developed and validated. The following compounds were included: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone. Individual stable isotope-labeled analogues were used as internal standards. Sample preparation was performed by liquid–liquid extraction, followed by oxime derivatization to improve the ionization efficiency of the analytes. In contrast to the common derivatization-based methods, the reaction was incorporated into the sample preparation process and the only additional step due to the derivatization was a short heating of the autosampler vials before the sample injection. Chromatographic separation was achieved on a reversed-phase column using a methanol–water gradient. For the analyte detection, a triple quadrupole instrument with electrospray ionization was used. Total run time was 7.0 min and the lower limits of quantification were in the range of 0.03–0.34 nM (0.01–0.10 ng/ml), depending on the analyte. The method was validated using human serum samples from both sexes and applied for the serum steroid profiling of endometriosis patients.

► LC–MS/MS method for seven human serum steroids was developed.
► Oxime derivatization used for increased method sensitivity.
► Total run time 7 min with quantification limits between 0.03 and 0.34 nM.
► Method was validated and used for serum steroid profiling of endometriosis patients.